PROJECT SUMMARY Asthma is a highly prevalent chronic inflammatory airway disease with tight genetic and mechanistic links to allergy. Hyperproduction of antibodies of the IgE isotype is a hallmark feature of allergy and the ?Th2 high? allergic asthma endotype. IgE is produced when B cells undergo class switch recombination (CSR) and differentiate into antibody secreting plasma cells. Secreted IgE arms mast cells and basophils to release inflammatory mediators in response to allergen exposure, fueling both acute anaphylactic responses and chronic inflammation. A therapeutic antibody targeting IgE reduces asthma symptoms and exacerbations in many patients with moderate to severe persistent disease. Understanding the molecular programming that underlies IgE production may lead to novel and more effective approaches for the prevention and treatment of allergy and asthma. MicroRNAs (miRNAs) are tiny regulators of gene expression that mediate powerful biological effects through their concerted action on networks of target mRNAs. Over 100 distinct miRNAs are expressed in B cells, but critical physiological functions have been assigned to few, so far. Several years ago, we developed a robust screening platform for uncovering miRNA regulators of helper T cell differentiation. We have now fully adapted this system for use in primary B cells and used it to uncover miRNA regulators of CSR and IgE production, including miR-221/222, miR-155 and several other strong novel candidates for further study. The central objective of this proposal is to leverage B cell miRNA:target networks to discover genes and pathways involved in the development and pathogenesis of allergy and asthma. Guided by strong preliminary data, we will rigorously interrogate the function of miR-221/222 and select other miRNA families implicated in IgE production using in vitro culture systems and mouse aeroallergen exposure models of asthma. We will perform comparative Ago2 HITS-CLIP on B cells and combine this biochemical approach with gene expression and computational analyses to generate a map of experimentally defined functional miRNA binding sites throughout the B cell transcriptome. These data will facilitate miRNA-directed discovery of genes and pathways involved in IgE production. Based on preliminary data, we have selected the transcription factor Foxp1 and two other novel miR-221/222 targets to be the first subjects of detailed analysis, taking advantage of existing mutant mice, small molecule inhibitors, and CRISPR/Cas9 genome editing protocols in mouse and human primary B cells. We expect the proposed research to generate novel insights about allergic sensitization and the immunopathogenesis of allergic asthma. In addition, our studies will advance our fundamental understanding of the network properties of miRNA regulation of gene expression and cell behavior.